Cloning and expression of bacillus thuringiensis gene toxic to beetles of the order coleoptera

ABSTRACT

The toxin gene encoding a protein toxic to beetles of the order Coleoptera, named M-7, has been cloned and expressed. M-7 is a novel Bacillus thuringiensis strain which has been deposited with a recognized culture repository.

This application is a continuation of U.S. application Ser. No. 282,143, filed Dec. 9, 1988, now abandoned, which is a division of U.S. application Ser. No. 767,227, filed Aug. 16, 1985 now U.S. Pat. No. 4,865,981.

BACKGROUND OF THE INVENTION

The spore-forming microorganism Bacillus thuringiensis (Bt) produces the best-known insect toxin. The toxin is a protein, designated as δ-endotoxin. It is synthesized by the Bt sporulating cell. The toxin, upon being ingested in its crystalline form by susceptible insect larvae, is transformed into biologically active moieties by the insect gut juice proteases. The primary target is insect cells of the gut epithelium, which are rapidly destroyed. Experience has shown that the activity of the Bt toxin is so high that only nanogram amounts are required to kill susceptible insect larvae.

The reported activity spectrum of Bt covers insect species within the order Lepidoptera, which is a major insect problem in agriculture and forestry. The activity spectrum also includes the insect order Diptera, wherein reside some species of mosquitoes and blackflies. See Couch, T. L., (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-67: Beegle, C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology, 20:97-104.

Krieg et al., Z. ang. Ent. (1983) 96:500-508, describe a Bt isolate named Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles of the order Coleoptera. These are Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni. This is the only known Bt isolate reported to contain such activity; all previously identified Bt strains have had activity against caterpillars (order Lepidoptera) or larvae of certain flies (order Diptera).

The Krieg et al. Bt isolate is not available for side-by-side comparison with the Bt isolate used as the source of the novel Bt gene of the subject invention. Therefore, since the Krieg et al. Bt isolate is not available to the public, the Krieg et al. publication is not a valid patent law reference under U.S. law.

BRIEF SUMMARY OF THE INVENTION

Disclosed and claimed is the cloning and expression of the toxin gene toxic to beetles of the order Coleoptera. The toxin produced by the cloned gene has activity against beetles of the order Coleoptera but not against Trichoplusia ni, Spodoptera exigua or Aedes aegypti. Included in the Coleoptera are various Diabrotica species (family Chrysomelidae) that are responsible for large agricultural losses. For example, D. undecimpunctata (western spotted cucumber beetle), D. longicornis (northern corn rootworm), D. virgitera (western corn rootworm), and D. undecimpunctata howardi (southern corn rootworm).

DETAILED DISCLOSURE OF THE INVENTION

The Bacillus thuringiensis isolate used as the source of the toxin gene of the subject invention, designated "M-7," is unusual in having a unique parasporal body (crystal) which under phase contrast microscopy is dark in appearance with a flat, square configuration.

A subculture of B. thuringiensis M-7 has been deposited in the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., USA on February 27, 1985. The culture was assigned the accession number NRRL B-15939 by the repository. This deposit is available to the public upon the grant of a patent disclosing it. The deposit is also available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

B. thuringiensis M-7, NRRL B-15939, can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by first separating the Bt spores and crystals from the fermentation broth by means well known in the art. The DNA (chromosomal and plasmid) from the cells can be isolated by standard procedures and purified by procedures well known in the art. For example, such standard procedures are disclosed in Maniatis et al. Molecular Cloning (1982) Cold Spring Harbor Laboratory.

The purified DNA then can be digested with a suitable restriction endonuclease.

A gene bank of B.t. M-7 DNA then can be constructed. In the subject invention, the purified B.t. M-7 DNA, obtained as described above, was digested with the restriction endonuclease BamHI and cloned into the BamHI site of the well-known and available plasmid pBR322.

Once the gene bank of B.t. M-7 DNA was constructed, it then became necessary to construct a DNA probe to screen the gene bank. The construction of this critical DNA probe was initiated by the isolation of M-7 toxin crystals from a culture of B.t. M-7.

The recovered M-7 toxin crystals were purified by standard procedures and then digested with trypsin to produce peptide fragments. The amino acid sequences of several of these tryptic fragments was determined by standard procedures. Subsequently, after selection of certain sequences, a probe was chemically synthesized by known means. The resulting probe was labelled and hybridized by procedures known in the art. The net result was the detection of positive clones, i.e., those that hybridized to the constructed probe.

A representative of the positive clones was subjected to a western blot using rabbit anti M-7 crystal antiserum developed by standard procedures. Confirmation of the success of the cloning and expression of M-7 toxin was obtained when a positive reaction was observed with the positive clone and the antibody against M-7 toxin crystal.

The recombinant plasmids isolated from representative positive clones were found to have a 5.8 kb DNA fragment inserted into the BamHI site. This 5.8 kb DNA fragment was excised from a representative positive clone (pCH-B3) with BamHI, purified, and then subcloned into the BamHI site of the known and available plasmid pRO1614 (J. Bact. [1982] 150:60, U.S. Pat. No. 4,374,200). Plasmid pRO1614 is available from the Northern Regional Research Laboratory, address below, where its deposit number is NRRL B-12127. The plasmid is derived from pBR322 and has unique HindIII, BamHI, and SalI and PvuII restriction sites; a PstI insertion includes the carbenicillin resistance gene and a P. aeruginosa replication system. Pseudomonas fluorescens was transformed with this constructed shuttle vector and the expression of M-7 toxin was verified by its identification on a western blot.

Plasmid pCH-B3, or plasmid pRO1614 with the 5.8 kb fragment insert, can be recovered from their bacterial hosts by well-known procedures, e.g., using the cleared lysate-isopycnic density gradient procedures. If desired, the 5.8 kb fragment can be exercised from pRO1614 by digestion with BamHI and cloned into a different vector for transformation into another host. These procedures are all well known to persons skilled in the art.

Plasmid pCH-B3, in an E. coli host, was deposited with the ARS Patent Collection, Culture Collection Research-Fermentation Laboratory, Northern Regional Research Center, Peoria, Ill. 61604. The deposit was made in the permanent collection of the repository to be maintained by the repository for at least 30 years. The deposit was made on July 18, 1985, and given the accession number NRRL B-15981. A subculture is available to the public upon the grant of a patent disclosing the deposit. The deposit is also available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

The toxin gene of the subject invention can be introduced into a wide variety of microbial hosts. Expression of the toxin gene (M-7) results, directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable hosts, e.g., Pseudomonas, the microbes can be applied to the situs of beetles of the order Coleoptera where they will proliferate and be ingested by the susceptible beetles. The result is a control of the unwanted beetles. Alternatively, the microbe hosting the toxin M-7 gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of target pest(s). The resulting product retains the toxicity of the M-7 toxin.

Where the M-7 toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera, Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing the M-7 gene expressing the toxin into the microorganism host under conditions which allow for stable maintenance and expression of the gene. One can provide for DNA constructs which include the transcriptional and translational regulatory signals for expression of the toxin gene, the toxin gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.

The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In some instances, it may be desirable to provide for regulative expression of the toxin, where expression of the toxin will occur only after release into the environment. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the microorganisms. For example, a temperature sensitive regulatory region may be employed, where the organisms may be grown up in the laboratory without expression of a toxin, but upon release into the environment, expression would begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the environment would allow for expression of the toxin. For translational initiation, a ribosomal binding site and an initiation codon will be present.

Various manipulations may be employed for enhancing the expression of the messenger, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the messenger RNA. The initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construct or may be combined as a separate DNA fragment with the toxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selection of those hosts which have been modified or transformed. The marker will normally provide for selective advantage, for example, providing for biocide resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototrophy to an auxotrophic host, or the like. Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. One or more markers may be employed in the development of the constructs, as well as for modifying the host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. For example, genes expressing metal chelating agents, e.g., siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the toxin producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment of the vegetation to be protected.

Where no functional replication system is present, the construct will also include a sequence of at least 50 bp, preferably at lest about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the toxin gene will be in close proximity to the gene providing for the complementation as well as the gene providing for the competitive advantage. Therefore, in the event that the toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct.

A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the lambda left and right promoters, the Tac promoter, the naturally-occurring promoters associated with the toxin gene, where functional in the host. See for example, U.S. Pat. Nos. 4,332,898; 4,342,832 and 4,356,270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host. The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus which is stable in the host. A large number of plasmids are available, such as pBR322, pACYC184, RSF1010, pRO1614, and the like. See for example, Olson et al., (1982) J. Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, and U.S. Pat. Nos. 4,356,270; 4,362,817; and 4,371,625.

The M-7 gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against ummodified organisms or transferring organisms, when present. The transformants then can be tested for pesticidal activity.

Preferred hosts, particularly those in the phytosphere, will have certain characteristics which enhance the environmental stability of the toxins in the host. Protective qualities include a low level of proteolytic degradation, thick cell walls, pigmentation, and the like. Other characteristics of interest for the host include leaf affinity, lack of mammalian toxicity, attractiveness to pests for ingestion, ease of handling and storage, rate of proliferation in the field, competitiveness, and the like.

In the field applications, the transformant strain will be applied to its natural habitat, such as the rhizosphere or phylloplane of the plant to be protected from the pest. The transformant strain will grow in its natural habitat, while producing the M-7 toxin which will be absorbed and/or ingested by the larvae or adult pest, or have a toxic effect on the ova. The persistence of the microorganisms will provide for long-term protection of the vegetation, although repetitive administrations may be required from time to time. The organism may be applied by spraying, soaking, injection into the soil, seed coating, seedling coating or spraying, or the like. Where administered in the field, generally concentrations of the organism will be from 10⁶ to 10¹⁰ cells/ml, and the volume applied per hectare will be generally from about 0.1 oz to 2 lbs or more. Where administered to a plant part, the concentration of the organism will usually be from 10³ to 10⁶ cells/cm².

Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell when the then dead cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the M-7 gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.

The cell will usually be intact and be substantially in the proliferative form when killed, rather than in a spore form, although in some instances spores may be employed.

The cells may be inhibited from proliferation in a variety of ways, so long as the technique does not deleteriously affect the properties of the pesticide, nor diminish the cellular capability in protecting the pesticide. The techniques may involve physical treatment, chemical treatment, changing the physical character of the cell or leaving the physical character of the cell substantially intact, or the like.

Various techniques for inactivating the host cells include heat, usually 50° C. to 70° C.; freezing; UV irradiation; lyophilization; toxins, e.g., antibiotics; phenols; anilides, e.g., carbanilide and salicylanilide; hydroxyurea; quaternaries; alcohols; antibacterial dyes; EDTA and amidines; non-specific organic and inorganic chemicals, such as halogenating agents, e.g., chlorinating, brominating or iodinating agents; aldehydes, e.g., glutaraldehyde or formaldehyde; toxic gases, such as ozone and ethylene oxide; peroxide; psoralens; desiccating agents or the like, which may be used individually or in combination. The choice of agent will depend upon the particular pesticide, the nature of the host cell, the nature of the modification of the cellular structure, such as fixing and preserving the cell wall with crosslinking agents, or the like.

The cells generally will have enhanced structural stability which will enhance resistance to environmental degradation in the field. Where the pesticide is in a proform, the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bioavailability or bioactivity of the toxin.

The cellular host containing the M-7 pesticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the M-7 gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be fixed prior to harvesting.

The method of treating the host organism containing the toxin can fulfill a number of functions. First, it may enhance structural integrity. Second, it may provide for enhanced proteolytic stability of the toxin, by modifying the toxin so as to reduce its susceptibility to proteolytic degradation and/or by reducing the proteolytic activity of proteases naturally present in the cell. The cells are preferably modified at an intact stage and when there has been a substantial buildup of the toxin protein. These modifications can be achieved in a variety of ways, such as by using chemical reagents having a broad spectrum of chemical reactivity. The intact cells can be combined with a liquid reagent medium containing the chemical reagents, with or without agitation, at temperatures in the range of about -10° to 60° C. The reaction time may be determined empirically and will vary widely with the reagents and reaction conditions. Cell concentrations will vary from about 10² to 10¹⁰ per ml.

Of particular interest as chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (see Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of the target pest(s).

For halogenation with iodine, temperatures will generally range from about 0° to 50° C., but the reaction can be conveniently carried out at room temperature. Conveniently, the iodination may be performed using triiodide or iodine at 0.5 to 5% in an acidic aqueous medium, particularly an aqueous carboxylic acid solution that may vary from about 0.5-5M. Conveniently, acetic acid may be used, although other carboxylic acids, generally of from about 1 to 4 carbon atoms, may also be employed. The time for the reaction will generally range from less than a minute to about 24 hr, usually from about 1 to 6 hr. Any residual iodine may be removed by reaction with a reducing agent, such as dithionite, sodium thiosulfate, or other reducing agent compatible with ultimate usage in the field. In addition, the modified cells may be subjected to further treatment, such as washing to remove all of the reaction medium, isolation in dry form, and formulation with typical stickers, spreaders, and adjuvants generally utilized in agricultural applications, as is well known to those skilled in the art.

Of particular interest are reagent capable of crosslinking the cell wall. A number of reagents are known in the art for this purpose. The treatment should result in enhanced stability of the pesticide. That is, there should be enhanced persistence or residual activity of the pesticide under field conditions. Thus, under conditions where the pesticidal activity of untreated cells diminishes, the activity of treated cells remains for periods of from 1 to 3 times longer.

The cells may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueousbased or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-16% by weight of the solids in the liquid phase. The formulations will generally have from about 10² to about 10⁴ cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling or the like.

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Culturing B. thuringiensis M-7 NRRL B-15939

A subculture or starter culture of B. thuringiensis M-7 NRRL B-15939 can be used to inoculate the following medium, known as LB broth:

    ______________________________________                                         Tryptone       10            gm                                                Yeast extract  5             gm                                                NaCl           5             gm                                                5N NaOH        0.6           ml                                                Water          1000          ml                                                ______________________________________                                    

As per standard microbiological techniques, the above medium would be sterilized prior to inoculation and the inoculations would be done using aseptic procedures. The M-7 cells are grown for 3-4 days at 30° C.

A detailed procedure is as follows:

A series of 150 ml Erlenmeyer flasks containing sterile PWYE medium (peptone 5.0%; yeast extract 0.1%; NaCl, 0.5% in 1 liter of water; adjust pH to 7.5) are inoculated from a petri plate culture of B. thuringiensis M-7, NRRL B-15939. The flasks are incubated at 30° C. on a rotary shaker (200 rpm) overnight. From this starter culture, 300 ml of LB broth in a 2-liter flask is inoculated using 7.5 ml of the starter. The LB-broth flasks are incubated under the same conditions as the starter, but are harvested after 4 days.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The Bt spores and crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.

EXAMPLE 2 Cloning and Expression of M-7 Toxin Gene

Total DNA (chromosomal and plasmid) was isolated from the M-7 cells of Example 1 and purified by standard procedures. The resulting purified DNA was digested with the restriction endonuclease BamHI, using the supplier's instruction. The digested DNA was then cloned into the BamHI site of the well-known plasmid pBR322 to give a gene bank of M-7 DNA. This cloning procedure was done following standard well-known procedures.

A DNA probe to screen the gene bank was obtained as follows: M-7 crystals were isolated from a culture grown in NYSM medium (10 gm tryptone, 5 gm NaCl, 5 gm yeast extract, 2 gm MgSO₄. 7H₂ O, 1000 ml water, pH 7.5) overnight at 30° C. The purified crystals were dissolved in 8 M urea, 0.1 M glycine, pH 8.2 and digested with trypsin overnight at room temperature. The resulting peptide fragments were separated on a C₄ reverse phase high pore column with a 180 min gradient of 91% solution A (0.1% trifluoroacetic acid in H₂ O) to 40% solution A in 0.1% trifluoroacetic acid in acetonitrile. The aminoacid sequences of several tryptic fragments were obtained and a sequence of 6 aminoacids were selected for synthesis of a mixed probe, 17 bases in length, with a redundancy of 32.

The probe was end-labeled with polynucleotide kinase and [γ-³² P]ATP and hybridized to bacterial colonies containing recombinant plasmids as constructed for the M-7 gene bank. The colony filters were prepared according to Hanahan and Meselson (1980) Gene 10:63-67. Positive colonies were identified by autoradiography. The recombinant plasmids isolated from seven positive clones (pCH-B3 as representative) were found to have a 5.8 kb (kilobase pairs) DNA fragment inserted into the BamHI site.

A western blot (Burnette, W. N. [1981] Anal. Biochem. 112:195) of pCH-B3 was performed on an SDS-PAGE of an overnight culture, using rabbit anti-M-7 crystal antiserum. A protein of about 86 kilodalton was identified. The clone pCH-B3, therefore, contains an M-7 DNA fragment that encodes for a protein having serological identity with the protein from the M-7 crystals. The recombinant protein may be bigger than the toxin from solubilized M-7 crystals because of unavailability of transcriptional and/or translational stop signals in the given plasmid construction.

EXAMPLE 3 Production of M-7 Toxin Protein by Clone pCH-B3

A 20 liter culture of pCH-B3 (L-broth with 70 μg/ml Ampicillin) was grown in a fermenter and harvested at OD600=3.35. The cell pellet was washed with water and resuspended in 500 ml glycine buffer (0.1 M glycine, pH 8.0 with tris base) containing 2 g lysozyme, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM TPCK (1-tosylamide-2-phenyl ethylchloromethyl ketone), and 500 μg DNase I and incubated at room temperature for 30 min. The pH was then raised to 10 with NaOH and the cells were further ruptured in a bead beater (Biospec Products, Bartlesville, OK) on ice with four 30 second bursts 5 min apart. The extract was then centrifuged at 10,000 x g for 30 min.

EXAMPLE 4 Isolation and Purification of M-7 Toxin Protein Produced by Clone pCH-B3

The protein from pCH-B3 was purified using affinity chromatography (Cuatrecasa, P. and Anfinsen, C. B. [1971] Meth. Enzymology Vol. 22 [ed. W. B. Jacoby] Acad. Press, N.Y.) as follows: Sepharose was activated with cyanogen bromide as described by Cuatrecasa and Anfinsen. Rabbit anti-M-7 crystal serum was added to the activated Sepharose and incubated overnight at room temperature with constant agitation. The affinity resign was then washed with 1% ethanolamine, 3 M NaCl, pH 9.2, and then with TBS (0.02 M tris-HCl, 0.07 M NaCl, pH 7.5) containing 0.02% sodium azide. The column was equilibrated in 0.1 M glycine pH 10 (with tris base) containing 1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM PMSF, 1 mM TPCK, and 0.02% sodium azide. The E. coli extract, prepared above, was loaded onto the column and recirculated for 64 hr at 4° C. The extract was washed from the column with 1 M NaCl and 0.1 M glycine-tris pH 10, and the bound M-7 toxin was removed from the column with 3 M sodium perchlorate, 0.1 M glycine-tris pH 10. The M-7 toxin was then dialyzed against water and concentrated (MicroPro D: Con, Pierce Chem. Co., Rockford, Ill.).

The purified M-7 toxin can be administered (applied) to vegetation susceptible to infestation by beetles of the order Coleoptera to protect the vegetation. Advantageously, the M-7 toxin will be made environmentally stable by use of suitable coatings well known to persons skilled in the art.

EXAMPLE 5 Subcloning and Expression of M-7 Toxin Gene into Pseudomonas fluorescens

The 5.8 kb DNA fragment carrying the M-7 toxin gene was excised from plasmid pCH-B3 with BamHI, purified, and subcloned into the BamHI site of the plasmid pR01614. Pseudomonas fluorescens was transformed with this plasmid. The expression of M-7 toxin by recombinant Pseudomonas cells was verified by its identification on a western blot.

EXAMPLE 6 Testing of B. thuringiensis M-7 NRRL B-15939 Spores and Crystal

B. thuringiensis M-7 NRRL B-15939 spores and crystal, obtained as described above, were tested against various insects. The insect species tested and a summary of the results are listed below in Table 1.

                                      TABLE 1                                      __________________________________________________________________________     Insects Evaluated for Susceptibility to Bacillus thuringiensis Isolate         M-7                                                                            Order  Family  Species  Common Name                                                                              Stages Tested                                                                         Activity                              __________________________________________________________________________     Coleoptera                                                                            Chrysomelidae                                                                          Diabrotica                                                                              Western spotted                                                                          Adult, larva                                                                          +                                                    undecimpunctata                                                                         cucumber beetle                                                       Pyrrhalta                                                                               Elm leaf beetle                                                                          Adult, larva                                                                          ++++                                                 luteola                                                                        Haltica    --      Adult, larva                                                                          +++                                                  tombacina                                                              Curculionidae                                                                          Otiorhynchus                                                                            Black vine weevil                                                                        Larva  ++                                                   sulcatus                                                               Tenebrionidae                                                                          Tenebrio Yellow mealworm                                                                          Larva  ++                                                   molitor                                                                        Tribolium                                                                               Red flour beetle                                                                         Adult, larva                                                                          -                                                    castaneum                                                              Dermestidae                                                                            Attagenus                                                                                 --      Larva  -                                                    megatoma                                                               Ptinidae                                                                               Gibbium    --      Adult  -                                                    psylloides                                                      Diptera                                                                               Culicidae                                                                              Aedes    Yellow fever                                                                             Larva  -                                                    aegypti  mosquito                                               Lepidoptera                                                                           Noctuidae                                                                              Spodoptera                                                                              Beet armyworm                                                                            Larva  -                                                    exigua                                                                         Trichoplusia                                                                            Cabbage looper                                                                           Larva  -                                                    ni                                                              __________________________________________________________________________

The method used to test for D. undecimpunctata (WSCB) activity consisted of spraying a spore/crystal suspension onto leaf discs of lettuce in a spray tower apparatus. (The larvae of this species are reared on lettuce leaves.) The spray was dried in a laminar flow hood and placed in a container on moist filter paper. Ten larvae of WSCB were added and the containers were incubated at 25° C. and 14 hr photoperiod. Fresh treated discs were added as needed. Inhibition of feeding was noted and mortality was recorded at 5 and 7 days. Results of 2 bioassays are given in Table 2.

                  TABLE 2                                                          ______________________________________                                         Results of 2 Bioassays of Bacillus thuringiensis                               M-7 Against Second Instar Diabrotica undecimpunctata U.                        at 7 Days Post-Inoculation                                                                    Avg. no.                                                                       leaf discs                                                      Treatment      consumed/rep.                                                                              % Mortality                                         ______________________________________                                         Exp 1                                                                          Control        3            7.5 ± 15.0                                      4.3 × 10.sup.7 spores/ml                                                                <1          27.5 ± 9.6                                       4.3 × 10.sup.8 spores/ml                                                                0           62.5 ± 26.3                                      Exp 2                                                                          Control        1           12.5 ± 12.6                                      1 × 10.sup.6 spores/ml                                                                  <1          30.0 ± 8.2                                       1 × 10.sup.7 spores/ml                                                                  0           50.0 ± 21.6                                      ______________________________________                                    

In order to test the M-7 toxin for activity against Pyrrhalta luteola (elm leaf beetle), a suspension of solubilized protein from M-7 crystals was applied to elm leaves. The dried leaves were then placed in a container on moist sand. Five to ten larvae of P. luteola were added and the containers were incubated at room temperature. Mortality was recorded at 3 and 5 days. An LC₅₀ of 120 ng toxin/cm² of leaf surface was calculated from these assays. 

We claim:
 1. A live Pseudomonas fluorescens capable of proliferating in the phytosphere of vegetation, wherein said microorganism contains a 5.8 kb DNA fragment from Bacillus thuringiensis M-7, said fragment comprising a gene encoding a toxin toxic to beetles of the order Coleoptera expressed in said microorganism wherein said toxin is maintained at a biocidal level in said microorganism, so that upon ingestion by said pest of said microorganism in normal feeding, a biocidal level is ingested.
 2. A live Pseudomonas fluorescens capable of colonizing the phytosphere of vegetation wherein said microorganism contains a Bacillus thuringiensis gene encoding a toxin toxic to beetles of the order Coleoptera expressed in said microorganism, and wherein said toxin is maintained at a level in said microorganism, so that upon ingestion by said pest of said microorganism in normal feeding, a growth-inhibiting level is ingested, and wherein said Bacillus thuringiensis toxin gene DNA is as follows:

    __________________________________________________________________________                                         ATGA                                                                               ATCCGAACAA                             TCGAAGTGAA                                                                               CATGATACAA                                                                               TAAAAACTAC                                                                               TGAAAATAAT                                                                               GAGGTGCCAA                             CTAACCATGT                                                                               TCAATATCCT                                                                               TTAGCGGAAA                                                                               CTCCAAATCC                                                                               AACACTAGAA                             GATTTAAATT                                                                               ATAAAGAGTT                                                                               TTTAAGAATG                                                                               ACTGCAGATA                                                                               ATAATACGGA                             AGCACTAGAT                                                                               AGCTCTACAA                                                                               CAAAAGATGT                                                                               CATTCAAAAA                                                                               GGCATTTCCG                             TAGTAGGTGA                                                                               TCTCCTAGGC                                                                               GTAGTAGGTT                                                                               TCCCGTTTGG                                                                               TGGAGCGCTT                             GTTTCGTTTT                                                                               ATACAAACTT                                                                               TTTAAATACT                                                                               ATTTGGCCAA                                                                               GTGAAGACCC                             GTGGAAGGCT                                                                               TTTATGGAAC                                                                               AAGTAGAAGC                                                                               ATTGATGGAT                                                                               CAGAAAATAG                             CTGATTATGC                                                                               AAAAAATAAA                                                                               GCTCTTGCAG                                                                               AGTTACAGGG                                                                               CCTTCAAAAT                             AATGTCGAAG                                                                               ATTATGTGAG                                                                               TGCATTGAGT                                                                               TCATGGCAAA                                                                               AAAATCCTGT                             GAGTTCACGA                                                                               AATCCACATA                                                                               GCCAGGGGCG                                                                               GATAAGAGAG                                                                               CTGTTTTCTC                             AAGCAGAAAG                                                                               TCATTTTCGT                                                                               AATTCAATGC                                                                               CTTCGTTTGC                                                                               AATTTCTGGA                             TACGAGGTTC                                                                               TATTTCTAAC                                                                               AACATATGCA                                                                               CAAGCTGCCA                                                                               ACACACATTT                             ATTTTTACTA                                                                               AAAGACGCTC                                                                               AAATTTATGG                                                                               AGAAGAATGG                                                                               GGATACGAAA                             AAGAAGATAT                                                                               TGCTGAATTT                                                                               TATAAAAGAC                                                                               AACTAAAACT                                                                               TACGCAAGAA                             TATACTGACC                                                                               ATTGTGTCAA                                                                               ATGGTATAAT                                                                               GTTGGATTAG                                                                               ATAAATTAAG                             AGGTTCATCT                                                                               TATGAATCTT                                                                               GGGTAAACTT                                                                               TAACCGTTAT                                                                               CGCAGAGAGA                             TGACATTAAC                                                                               AGTATTAGAT                                                                               TTAATTGCAC                                                                               TATTTCCATT                                                                               GTATGATGTT                             CGGCTATACC                                                                               CAAAAGAAGT                                                                               TAAAACCGAA                                                                               TTAACAAGAG                                                                               ACGTTTTAAC                             AGATCCAATT                                                                               GTCGGAGTCA                                                                               ACAACCTTAG                                                                               GGGCTATGGA                                                                               ACAACCTTCT                             CTAATATAGA                                                                               AAATTATATT                                                                               CGAAAACCAC                                                                               ATCTATTTGA                                                                               CTATCTGCAT                             AGAATTCAAT                                                                               TTCACACGCG                                                                               GTTCCAACCA                                                                               GGATATTATG                                                                               GAAATGACTC                             TTTCAATTAT                                                                               TGGTCCGGTA                                                                               ATTATGTTTC                                                                               AACTAGACCA                                                                               AGCATAGGAT                             CAAATGATAT                                                                               AATCACATCT                                                                               CCATTCTATG                                                                               GAAATAAATC                                                                               CAGTGAACCT                             GTACAAAATT                                                                               TAGAATTTAA                                                                               TGGAGAAAAA                                                                               GTCTATAGAG                                                                               CCGTAGCAAA                             TACAAATCTT                                                                               GCGGTCTGGC                                                                               CGTCCGCTGT                                                                               ATATTCAGGT                                                                               GTTACAAAAG                             TGGAATTTAG                                                                               CCAATATAAT                                                                               GATCAAACAG                                                                               ATGAAGCAAG                                                                               TACACAAACG                             TACGACTCAA                                                                               AAAGAAATGT                                                                               TGGCGCGGTC                                                                               AGCTGGGATT                                                                               CTATCGATCA                             ATTGCCTCCA                                                                               GAAACAACAG                                                                               ATGAACCTCT                                                                               AGAAAAGGGA                                                                               TATAGCCATC                             AACTCAATTA                                                                               TGTAATGTGC                                                                               TTTTTAATGC                                                                               AGGGTAGTAG                                                                               AGGAACAATC                             CCAGTGTTAA                                                                               CTTGGACACA                                                                               TAAAAGTGTA                                                                               GACTTTTTTA                                                                               ACATGATTGA                             TTCGAAAAAA                                                                               ATTACACAAC                                                                               TTCCGTTAGT                                                                               AAAGGCATAT                                                                               AAGTTACAAT                             CTGGTGCTTC                                                                               CGTTGTCGCA                                                                               GGTCCTAGGT                                                                               TTACAGGAGG                                                                               AGATATCATT                             CAATGCACAG                                                                               AAAATGGAAG                                                                               TGCGGCAACT                                                                               ATTTACGTTA                                                                               CACCGGATGT                             GTCGTACTCT                                                                               CAAAAATATC                                                                               GAGCTAGAAT                                                                               TCATTATGCT                                                                               TCTACATCTC                             AGATAACATT                                                                               TACACTCAGT                                                                               TTAGACGGGG                                                                               CACCATTTAA                                                                               TCAATACTAT                             TTCGATAAAA                                                                               CGATAAATAA                                                                               AGGAGACACA                                                                               TTAACGTATA                                                                               ATTCATTTAA                             TTTAGCAAGT                                                                               TTCAGCACAC                                                                               CATTCGAATT                                                                               ATCAGGGAAT                                                                               AACTTACAAA                             TAGGCGTCAC                                                                               AGGATTAAGT                                                                               GCTGGAGATA                                                                               AAGTTTATAT                                                                               AGACAAAATT                             GAATTTATTC                                                                               CAGTGAAT                                                             __________________________________________________________________________

and equivalent nucleotide sequences coding for molecules with the following amino acid sequence:

    __________________________________________________________________________                  5               10             15              20                 Met                                                                               Asn                                                                               Pro                                                                               Asn                                                                               Asn Arg                                                                               Ser                                                                               Glu                                                                               His                                                                               Asp                                                                               Thr                                                                               Ile                                                                               Lys                                                                               Thr                                                                               Thr Glu                                                                               Asn                                                                               Asn                                                                               Glu Val                             25              30             35               40                Pro                                                                               Thr                                                                               Asn                                                                               His                                                                               Val Gln                                                                               Tyr                                                                               Pro                                                                               Leu                                                                               Ala                                                                               Glu                                                                               Thr                                                                               Pro                                                                               Asn                                                                               Pro Thr                                                                               Leu                                                                               Glu                                                                               Asp Leu                             45              50             55               60                Asn                                                                               Tyr                                                                               Lys                                                                               Glu                                                                               Phe Leu                                                                               Arg                                                                               Met                                                                               Thr                                                                               Ala                                                                               Asp                                                                               Asn                                                                               Asn                                                                               Thr                                                                               Glu Ala                                                                               Leu                                                                               Asp                                                                               Ser Ser                             65              70             75               80                Thr                                                                               Thr                                                                               Lys                                                                               Asp                                                                               Val Ile                                                                               Gln                                                                               Lys                                                                               Gly                                                                               Ile                                                                               Ser                                                                               Val                                                                               Val                                                                               Gly                                                                               Asp Leu                                                                               Leu                                                                               Gly                                                                               Val Val                             85              90             95              100                Gly                                                                               Phe                                                                               Pro                                                                               Phe                                                                               Gly Gly                                                                               Ala                                                                               Leu                                                                               Val                                                                               Ser                                                                               Phe                                                                               Tyr                                                                               Thr                                                                               Asn                                                                               Phe Leu                                                                               Asn                                                                               Thr                                                                               Ile Trp                            105             110            115              120                Pro                                                                               Ser                                                                               Glu                                                                               Asp                                                                               Pro Trp                                                                               Lys                                                                               Ala                                                                               Phe                                                                               Met                                                                               Glu                                                                               Gln                                                                               Val                                                                               Glu                                                                               Ala Leu                                                                               Met                                                                               Asp                                                                               Gln Lys                            125             130            135              140                Ile                                                                               Ala                                                                               Asp                                                                               Tyr                                                                               Ala Lys                                                                               Asn                                                                               Lys                                                                               Ala                                                                               Leu                                                                               Ala                                                                               Glu                                                                               Leu                                                                               Gln                                                                               Gly Leu                                                                               Gln                                                                               Asn                                                                               Asn Val                            145             150            155              160                Glu                                                                               Asp                                                                               Tyr                                                                               Val                                                                               Ser Ala                                                                               Leu                                                                               Ser                                                                               Ser                                                                               Trp                                                                               Gln                                                                               Lys                                                                               Asn                                                                               Pro                                                                               Val Ser                                                                               Ser                                                                               Arg                                                                               Asn Pro                            165             170            175              180                His                                                                               Ser                                                                               Gln                                                                               Gly                                                                               Arg Ile                                                                               Arg                                                                               Glu                                                                               Leu                                                                               Phe                                                                               Ser                                                                               Gln                                                                               Ala                                                                               Glu                                                                               Ser His                                                                               Phe                                                                               Arg                                                                               Asn Ser                            185             190            195              200                Met                                                                               Pro                                                                               Ser                                                                               Phe                                                                               Ala Ile                                                                               Ser                                                                               Gly                                                                               Tyr                                                                               Glu                                                                               Val                                                                               Leu                                                                               Phe                                                                               Leu                                                                               Thr Thr                                                                               Tyr                                                                               Ala                                                                               Gln Ala                            205             210            215              220                Ala                                                                               Asn                                                                               Thr                                                                               His                                                                               Leu Phe                                                                               Leu                                                                               Leu                                                                               Lys                                                                               Asp                                                                               Ala                                                                               Gln                                                                               Ile                                                                               Tyr                                                                               Gly Glu                                                                               Glu                                                                               Trp                                                                               Gly Tyr                            225             230            235              240                Glu                                                                               Lys                                                                               Glu                                                                               Asp                                                                               Ile Ala                                                                               Glu                                                                               Phe                                                                               Tyr                                                                               Lys                                                                               Arg                                                                               Gln                                                                               Leu                                                                               Lys                                                                               Leu Thr                                                                               Gln                                                                               Glu                                                                               Tyr Thr                            245             250            255              260                Asp                                                                               His                                                                               Cys                                                                               Val                                                                               Lys Trp                                                                               Tyr                                                                               Asn                                                                               Val                                                                               Gly                                                                               Leu                                                                               Asp                                                                               Lys                                                                               Leu                                                                               Arg Gly                                                                               Ser                                                                               Ser                                                                               Tyr Glu                            265             270            275              280                Ser                                                                               Trp                                                                               Val                                                                               Asn                                                                               Phe Asn                                                                               Arg                                                                               Tyr                                                                               Arg                                                                               Arg                                                                               Glu                                                                               Met                                                                               Thr                                                                               Leu                                                                               Thr Val                                                                               Leu                                                                               Asp                                                                               Leu Ile                            285             290            295              300                Ala                                                                               Leu                                                                               Phe                                                                               Pro                                                                               Leu Tyr                                                                               Asp                                                                               Val                                                                               Arg                                                                               Leu                                                                               Tyr                                                                               Pro                                                                               Lys                                                                               Glu                                                                               Val Lys                                                                               Thr                                                                               Glu                                                                               Leu Thr                            305             310            315              320                Arg                                                                               Asp                                                                               Val                                                                               Leu                                                                               Thr Asp                                                                               Pro                                                                               Ile                                                                               Val                                                                               Gly                                                                               Val                                                                               Asn                                                                               Asn                                                                               Leu                                                                               Arg Gly                                                                               Tyr                                                                               Gly                                                                               Thr Thr                            325             330            335              340                Phe                                                                               Ser                                                                               Asn                                                                               Ile                                                                               Glu Asn                                                                               Tyr                                                                               Ile                                                                               Arg                                                                               Lys                                                                               Pro                                                                               His                                                                               Leu                                                                               Phe                                                                               Asp Tyr                                                                               Leu                                                                               His                                                                               Arg Ile                            345             350            355              360                Gln                                                                               Phe                                                                               His                                                                               Thr                                                                               Arg Phe                                                                               Gln                                                                               Pro                                                                               Gly                                                                               Tyr                                                                               Tyr                                                                               Gly                                                                               Asn                                                                               Asp                                                                               Ser Phe                                                                               Asn                                                                               Tyr                                                                               Trp Ser                            365             370            375              380                Gly                                                                               Asn                                                                               Tyr                                                                               Val                                                                               Ser Thr                                                                               Arg                                                                               Pro                                                                               Ser                                                                               Ile                                                                               Gly                                                                               Ser                                                                               Asn                                                                               Asp                                                                               Ile Ile                                                                               Thr                                                                               Ser                                                                               Pro Phe                            385             390            395              400                Tyr                                                                               Gly                                                                               Asn                                                                               Lys                                                                               Ser Ser                                                                               Glu                                                                               Pro                                                                               Val                                                                               Gln                                                                               Asn                                                                               Leu                                                                               Glu                                                                               Phe                                                                               Asn Gly                                                                               Glu                                                                               Lys                                                                               Val Tyr                            405             410            415              420                Arg                                                                               Ala                                                                               Val                                                                               Ala                                                                               Asn Thr                                                                               Asn                                                                               Leu                                                                               Ala                                                                               Val                                                                               Trp                                                                               Pro                                                                               Ser                                                                               Ala                                                                               Val Tyr                                                                               Ser                                                                               Gly                                                                               Val Thr                            425             430            435              440                Lys                                                                               Val                                                                               Glu                                                                               Phe                                                                               Ser Gln                                                                               Tyr                                                                               Asn                                                                               Asp                                                                               Gln                                                                               Thr                                                                               Asp                                                                               Glu                                                                               Ala                                                                               Ser Thr                                                                               Gln                                                                               Thr                                                                               Tyr Asp                            445             450            455              460                Ser                                                                               Lys                                                                               Arg                                                                               Asn                                                                               Val Gly                                                                               Ala                                                                               Val                                                                               Ser                                                                               Trp                                                                               Asp                                                                               Ser                                                                               Ile                                                                               Asp                                                                               Gln Leu                                                                               Pro                                                                               Pro                                                                               Glu Thr                            465             470            475              480                Thr                                                                               Asp                                                                               Glu                                                                               Pro                                                                               Leu Glu                                                                               Lys                                                                               Gly                                                                               Tyr                                                                               Ser                                                                               His                                                                               Gln                                                                               Leu                                                                               Asn                                                                               Tyr Val                                                                               Met                                                                               Cys                                                                               Phe Leu                            485             490            495              500                Met                                                                               Gln                                                                               Gly                                                                               Ser                                                                               Arg Gly                                                                               Thr                                                                               Ile                                                                               Pro                                                                               Val                                                                               Leu                                                                               Thr                                                                               Trp                                                                               Thr                                                                               His Lys                                                                               Ser                                                                               Val                                                                               Asp Phe                            505             510            515              520                Phe                                                                               Asn                                                                               Met                                                                               Ile                                                                               Asp Ser                                                                               Lys                                                                               Lys                                                                               Ile                                                                               Thr                                                                               Gln                                                                               Leu                                                                               Pro                                                                               Leu                                                                               Val Lys                                                                               Ala                                                                               Tyr                                                                               Lys Leu                            525             530            535              540                Gln                                                                               Ser                                                                               Gly                                                                               Ala                                                                               Ser Val                                                                               Val                                                                               Ala                                                                               Gly                                                                               Pro                                                                               Arg                                                                               Phe                                                                               Thr                                                                               Gly                                                                               Gly Asp                                                                               Ile                                                                               Ile                                                                               Gln Cys                            545             550            555              560                Thr                                                                               Glu                                                                               Asn                                                                               Gly                                                                               Ser Ala                                                                               Ala                                                                               Thr                                                                               Ile                                                                               Tyr                                                                               Val                                                                               Thr                                                                               Pro                                                                               Asp                                                                               Val Ser                                                                               Tyr                                                                               Ser                                                                               Gln Lys                            565             570            575              580                Tyr                                                                               Arg                                                                               Ala                                                                               Arg                                                                               Ile His                                                                               Tyr                                                                               Ala                                                                               Ser                                                                               Thr                                                                               Ser                                                                               Gln                                                                               Ile                                                                               Thr                                                                               Phe Thr                                                                               Leu                                                                               Ser                                                                               Leu Asp                            585             590            595              600                Gly                                                                               Ala                                                                               Pro                                                                               Phe                                                                               Asn Gln                                                                               Tyr                                                                               Tyr                                                                               Phe                                                                               Asp                                                                               Lys                                                                               Thr                                                                               Ile                                                                               Asn                                                                               Lys Gly                                                                               Asp                                                                               Thr                                                                               Leu Thr                            605             610            615              620                Tyr                                                                               Asn                                                                               Ser                                                                               Phe                                                                               Asn Leu                                                                               Ala                                                                               Ser                                                                               Phe                                                                               Ser                                                                               Thr                                                                               Pro                                                                               Phe                                                                               Glu                                                                               Leu Ser                                                                               Gly                                                                               Asn                                                                               Asn Leu                            625             630            635              640                Gln                                                                               Ile                                                                               Gly                                                                               Val                                                                               Thr Gly                                                                               Leu                                                                               Ser                                                                               Ala                                                                               Gly                                                                               Asp                                                                               Lys                                                                               Val                                                                               Tyr                                                                               Ile Asp                                                                               Lys                                                                               Ile                                                                               Glu Phe                Ile                                                                               Pro                                                                               Val                                                                               Asn.                                                                  __________________________________________________________________________


3. A method for protecting vegetation from beetles of the order Coleoptera which comprises administering to the environment of said vegetation, a live Pseudomonas fluorescens according to claim
 1. 4. A method for protecting vegetation from beetles of the order Coleoptera which comprises administering to the environment of said vegetation, a live Pseudomonas fluorescens according to claim
 2. 5. A method for protecting vegetation from beetles of the order Coleoptera, which comprises applying to a plant part a live Pseudomonas fluorescens according to claims 3 or
 4. 6. A method according to claim 5, wherein said plant part is a seed. 